The integrity of nuclear signaling through receptor-dependent phosphorylation is critical for maintaining health. One protein that mediates transcriptional effects through cyclic-adenosine-3'-5'- monophosphate (cAMP)/Ca++-dependent pathways is the cAMP response element binding protein (CREB). Phosphorylation at serine 133 of the CREB protein has been shown to be essential for transcriptional activation of the somatostatin gene and other cAMP-responsive genes. However, the mechanism by which phosphorylation alters gene transcription is unknown. In this proposal, we carefully examine five possible mechanisms by which CREB phosphorylation can alter receptor-mediated gene transcription. Focusing on a single gene, the human proenkephalin gene, and a single protein, a rat CREB protein, we will determine if phosphorylation of CREB through either PKA or PKC alters the conformation of CREB, alters the self- association strength of CREB (most probably dimerization), alters the binding affinity of CREB for the nucleic acid template, alters the conformation of the CREB/nucleic acid complex and/or alters the association state of CREB with other proteins. We begin our analysis by utilizing a single form of purified CREB whose transcriptional effects are well characterized. Using this well-defined material, we will determine the site of PKA and PKC phosphorylation in vivo and in vitro by trypsin or tryp-K digestion, separation of peptides using capillary electrophoresis (CE) coupled to mass spectroscopy (MS) for sequencing. We will measure the effects of phosphorylation on CREB conformation by circular dichroism (CD). CD changes will be correlated with analysis of the CREB peptide fragments generated before and after phosphorylation by trypsin or tryp-K digestion. The size, kinetics and sequence of tryptic peptides will be compared before and after digestion using CE/MS. The conformational data will be used to evaluate the effect of phosphorylation on CREB's self-association constant that will be measured by calorimetry. The molecular weight of the self-associated state will be assessed by sedimentation equilibrium. We will measure the binding constant of CREB and phospho-CREB to the nucleic acid through filter binding analysis. We will examine, by fluorescence energy transfer, electron spectroscopic imaging and circular dichroism, if the conformation of the CREB binding site can adopt an alternative structure upon phosphorylation. By immobilizing CREB on columns, we will examine its association with other proteins from receptor-induced cell extracts. We will repeat all studies using purified CREB that is phosphorylated by either PKA or PKC. The results from this defined system will be used to identify the role for CREB phosphorylation in receptor-mediated alterations of gene expression. The data will be used to generate a more general understanding of the role of phosphorylation in cellular signaling.